Development of a DNA Microarray for Detection and Serotyping of Enterotoxigenic Escherichia coli

Enterotoxigenic Escherichia coli (ETEC) is a common pathogen worldwide causing infectious diarrhea, especially traveler''s diarrhea. Traditional physiological assays, immunoassays, and PCR-based methods for the detection of ETEC target the heat-labile enterotoxin and/or the heat-stable enterotoxin. Separate serotyping methods using antisera are required to determine the ETEC serogroup. In this study, we developed a DNA microarray that can simultaneously detect enterotoxin genes and the 19 most common O serogroup genes in ETEC strains. The specificity and reproducibility of this approach were verified by hybridization to 223 strains: 50 target reference or clinical strains and 173 other strains, including those belonging to other E. coli O serogroups and closely related species. The sensitivity of detection was determined to be 50 ng of genomic DNA or 10⁸ CFU per ml of organisms in pure culture. The random PCR strategy used in this study with minimal bias provides an effective alternative to multiplex PCR for the detection of pathogens using DNA microarrays. The assay holds promise for applications in the clinical diagnosis and epidemiological surveillance of pathogenic microorganisms.Enterotoxigenic Escherichia coli (ETEC) is the leading bacterial cause of infectious diarrhea in the developing world, causing infantile or cholera-like disease in all age groups (2). It is among the major etiologic agents, leading to an estimated 1.5 million deaths per year worldwide (13, 14). ETEC is also a major cause of traveler''s diarrhea (3, 8, 11) and the most common pathogen among the six recognized diarrheagenic categories of E. coli, especially in the developing world (18). ETEC strains produce one or both of the following two enterotoxins: heat-labile enterotoxin (LT) and heat-stable enterotoxin (ST). Two classes of STs—STa and STb—and two variants of STa—STp (initially discovered in isolates from pigs) and STh (initially discovered in isolates from humans)—have been described. The elt, estA, and estB genes encode the enterotoxins LT, STa, and STb, respectively (6, 23, 26).The O antigen comprises the outermost domain of the lipopolysaccharide molecule and is attached to the core oligosaccharide on the surfaces of Gram-negative bacteria (20). O antigens are among the most variable cellular constituents, imparting antigenic specificity. The composition of the O chain differs from strain to strain; more than 180 O-antigen structures are produced by different E. coli strains (25). The most common O serogroups reported in ETEC are O6, O8, O11, O15, O25, O27, O78, O85, O114, O115, O126, O128, O139, O148, O149, O159, O166, O167, and O173 (5, 18, 19, 31).Detection of ETEC has long relied on detection of the enterotoxins LT and/or ST by physiological assays and immunoassays, and serotyping has depended on assays using O-serogroup-specific antisera. These traditional approaches are slow and labor-intensive, and assays using antisera can be impeded by cross-reactivity. PCR assays, which are more rapid, sensitive, and specific, have also been widely used for ETEC diagnosis (15, 24). However, molecular methods for the serotyping of ETEC have not been developed.Molecular detection and typing by PCR and microarray techniques have many advantages over traditional methods. DNA microarrays provide an efficient approach for the parallel detection and analysis of a large number of pathogenic microorganisms. This technique has been applied to the detection of pathogens from all kinds of biological samples, including water, food, and soil (4, 7, 12, 17, 21).In this study, we developed a DNA microarray for the detection and typing of ETEC. The genes encoding the enterotoxins LT and ST were used for the detection of ETEC, and the serogroup-specific genes wzx and/or wzy were used for the typing of the 19 most common ETEC O serogroups. The microarray was examined for its specificity and sensitivity, and the findings of this study indicate that it is highly sensitive and reproducible.     

Genetic Fusions of Heat-Labile (LT) and Heat-Stable (ST) Toxoids of Porcine Enterotoxigenic Escherichia coli Elicit Neutralizing Anti-LT and Anti-STa antibodies

Enterotoxigenic Escherichia coli (ETEC) strains are a major cause of diarrheal disease in humans and farm animals. E. coli fimbriae, or colonization factor antigens (CFAs), and enterotoxins, including heat-labile enterotoxins (LT) and heat-stable enterotoxins (ST), are the key virulence factors in ETEC diarrhea. Unlike fimbriae or LT, STa has not often been included as an antigen in development of vaccines against ETEC diarrhea because of its poor immunogenicity. STa becomes immunogenic only after being coupled with a strongly immunogenic carrier protein. However, native or shorter STa antigens either had to retain toxic activity in order to become antigenic or elicited anti-STa antibodies that were not sufficiently protective. In this study, we genetically mutated the porcine LT (pLT) gene for a pLT_192(R→G) toxoid and the porcine STa (pSTa) gene for three full-length pSTa toxoids [STa_11(N→K), STa_12(P→F), and STa_13(A→Q)] and used the full-length pLT₁₉₂ as an adjuvant to carry the pSTa toxoid for pLT₁₉₂:pSTa-toxoid fusion antigens. Rabbits immunized with pLT₁₉₂:pSTa₁₂ or pLT₁₉₂:pSTa₁₃ fusion protein developed high titers of anti-LT and anti-STa antibodies. Furthermore, rabbit antiserum and antifecal antibodies were able to neutralize purified cholera toxin (CT) and STa toxin. In addition, preliminary data suggested that suckling piglets born by a sow immunized with the pLT₁₉₂:pSTa₁₃ fusion antigen were protected when challenged with an STa-positive ETEC strain. This study demonstrated that pSTa toxoids are antigenic when fused with a pLT toxoid and that the elicited anti-LT and anti-STa antibodies were protective. This fusion strategy could provide instructive information to develop effective toxoid vaccines against ETEC-associated diarrhea in animals and humans.Enterotoxigenic Escherichia coli (ETEC) strains, which colonize host small intestines and produce one or more enterotoxins, are the major cause of diarrheal disease in humans and farm animals. The virulence determinants of ETEC in diarrhea include fimbrial adhesins and enterotoxins (1, 6, 10, 27, 28, 37, 44, 48). Fimbrial adhesins mediate attachment of bacteria to host epithelium cells and facilitate subsequent bacteria colonization. Enterotoxins, including heat-stable enterotoxins (STa and STb) and heat-labile enterotoxins (LT) (19, 20, 35), disrupt intestinal fluid homeostasis and cause fluid and electrolyte hypersecretion through activation of adenyl cyclase (by LT) or guanylate cyclase (by STa) in small intestinal epithelial cells (21, 26). ETEC strains isolated from young pigs with diarrhea express LT, STa, STb, Stx2e, and enteroaggregative E. coli ST type 1 (EAST1), alone or combined (10, 15, 50). Recent experimental studies indicated that porcine ETEC strains expressing LT, STb, or STa alone are sufficiently virulent to cause diarrhea in young pigs (6, 48, 49).Porcine ETEC-associated diarrhea, especially postweaning diarrhea (PWD), causes a substantial economic loss to swine producers worldwide (18, 41). Currently, there are no vaccines available to effectively protect weaned pigs against ETEC infections. Experimental vaccines developed from fimbrial antigens alone showed only limited protection against ETEC strains (42). In addition, ETEC fimbriae are antigenically different. Thus, experimental vaccines developed from one specific fimbria could not provide protection against an ETEC strain expressing a different fimbria (42, 43). Moreover, recent evidence suggests that fimbriae may not function as protective antigens in the setting of naturally acquired infections and reinfections (7). Consequently, enterotoxin antigens have been reemphasized for ETEC vaccine development (43). Antitoxin vaccines currently under development largely use LT or its B subunit antigens because they are strongly immunogenic. The ST antigen cannot be used directly as a vaccine component because of its poor immunogenicity, unless it is coupled to a carrier protein and presented as a fusion or a chimeric protein (13, 22, 31, 38). Although a recent study suggested that anti-LT immunity may provide broader protection (14), experimental vaccine studies indicated that the induced anti-LT immunity provided protection only against LT-producing ETEC strains and not against ETEC strains that produce STa toxin (12, 13). As over two-thirds of human ETEC diarrhea cases and more than one-quarter of porcine ETEC diarrhea cases are caused by STa-producing ETEC strains (15, 16, 29, 30, 36, 45, 50), STa antigens must be included for developing broadly effective vaccines against ETEC infection.Porcine STa (pSTa), a protein that consists of 18 amino acids (human STa [hSTa] consists of 19 amino acids), is poorly immunogenic (35, 41). To include pSTa as a vaccine component, we need to enhance pSTa immunogenicity. In addition, a native pSTa is not suitable to be used in developing safe vaccines because it is sufficiently toxic to cause diarrhea. Therefore, we need to attenuate the pSTa toxicity. It has been reported that shorter synthetic hSTa peptides or hSTa with disulfide bonds disrupted showed toxicity reduction (4, 5, 19, 40, 46, 47). Moreover, several shorter synthetic hSTa peptides that had the 12th, 13th, or 14th amino acid residue replaced showed a great reduction in toxicity (46, 47). However, these shorter synthetic or disulfide bond-disrupted hSTa peptides either had not been characterized for immunogenicity or failed to induce protective immunity. Only when a shorter hSTa or an hSTa mutant (with disulfide bonds disrupted) was genetically fused to a carrier protein, such as the B subunit of cholera toxin (CT) or hLT, did the hSTa antigen become immunogenic (9, 31, 32, 33). However, anti-STa immunity from these fusion antigens was not sufficiently characterized, and retention of STa toxicity in these fusion proteins could cause safety concerns for their application in vaccine development (7, 9).No studies have been conducted to enhance pSTa immunogenicity and its potential application in vaccines against porcine ETEC infections. In this study, we mutated the porcine estA gene at nucleotides encoding the 11th, 12th, and 13th amino acids (which are homologous to the 12th, 13th, and 14th amino acids of hSTa) for three pSTa toxoids. These pSTa toxoids, which had all disulfide bonds retained, showed a great reduction in toxicity when examined in vitro (by cyclic GMP [cGMP] enzyme-linked immunosorbent assay [ELISA]) and in vivo (by porcine gut loop assay and challenge studies in a piglet model). We then genetically fused the mutated full-length porcine eltAB and estA genes for pLT₁₉₂:pSTa-toxoid fusion proteins. Purified toxoid fusion antigens were used to immunize adult rabbits to assess anti-LT and anti-STa antigenicity and antibody neutralization and to immunize a pregnant sow to preliminarily evaluate anti-STa immunity in protection against infection from an STa-producing ETEC strain.     

Genetic Fusions of Heat-Labile Toxoid (LT) and Heat-Stable Toxin b (STb) of Porcine Enterotoxigenic Escherichia coli Elicit Protective Anti-LT and Anti-STb Antibodies

Enterotoxigenic Escherichia coli (ETEC)-associated diarrhea causes a substantial economic loss to swine producers worldwide. The majority of ETEC strains causing porcine diarrhea, especially postweaning diarrhea (PWD), produce heat-labile toxin (LT) and heat-stable toxin b (STb). LT is commonly used in vaccine development, but STb has not been included because of its poor immunogenicity. As a virulence factor in porcine diarrhea, STb needs to be included as an antigen for development of broad-spectrum vaccines. In this study, we used an LT toxoid (LT_R192G [hereafter, LT₁₉₂]) derived from porcine ETEC to carry a mature STb peptide for LT₁₉₂-STb fusions to enhance STb immunogenicity for potential vaccine application. Anti-LT and anti-STb antibodies were detected in immunized rabbits and pigs. In addition, when challenged with an STb-positive ETEC strain, all 10 suckling piglets borne by immunized gilts remained healthy, whereas 7 out 9 piglets borne by unimmunized gilts developed moderate diarrhea. This study indicates that the LT₁₉₂-STb fusion enhanced anti-STb immunogenicity and suggests the LT₁₉₂-STb fusion antigen can be used in future vaccine development against porcine ETEC diarrhea.Enterotoxigenic Escherichia coli (ETEC) strains that produce heat-labile (LT) and heat-stable (ST) enterotoxins are a major cause of diarrheal disease (27, 32). Bacterial adhesins and enterotoxins are the virulence determinants in ETEC-associated diarrhea (1, 4, 19, 20, 26, 33, 34). Porcine ETEC-associated diarrhea, especially postweaning diarrhea (PWD), causes substantial economic loss to swine producers worldwide (15, 28). Currently, there are no effective vaccines available to protect young pigs against PWD. Antitoxin vaccines currently under development largely use LT antigens because they are strongly immunogenic, whereas STb antigens have not been included. However, STb is the toxin most commonly found in ETEC strains associated with PWD (36). Moreover, an ETEC strain expressing STb as the only toxin caused diarrhea in over half of the gnotobiotic pigs tested (34). Therefore, STb antigens need to be included for development of broadly effective vaccines against porcine diarrhea.The STb antigen cannot be used directly as a vaccine component because of the poor immunogenicity. Previous studies demonstrated that a small and poorly immunogenic molecule became more immunogenic when it was conjugated to a strongly immunogenic carrier protein (3, 8, 12, 13, 16, 22, 23, 37). A detoxified heat-labile toxin protein (hLT₁₉₂, where hLT₁₉₂ represents human-type LT_R192G) derived from the LT genes isolated from a human E. coli strain retains LT immunogenicity but has toxicity substantially reduced and has been commonly used as an antigen and/or an adjuvant in vaccine development against bacterial and viral pathogens. In this study, we used an analogous detoxified LT protein, designated LT₁₉₂, as the carrier to enhance STb immunogenicity. This LT₁₉₂ protein was produced by mutating the porcine-type LT genes (eltAB) isolated from a porcine E. coli strain. We fused the estB gene coding for the mature STb peptide to the mutated, full-length porcine-type LT₁₉₂ genes and examined LT₁₉₂-STb fusion proteins in enhancement of STb immunogenicity and potential vaccine application against porcine diarrhea.     

Variation in Susceptibility of Bloodstream Isolates of Candida glabrata to Fluconazole According to Patient Age and Geographic Location in the United States in 2001 to 2007

We examined the susceptibilities to fluconazole of 642 bloodstream infection (BSI) isolates of Candida glabrata and grouped the isolates by patient age and geographic location within the United States. Susceptibility of C. glabrata to fluconazole was lowest in the northeast region (46%) and was highest in the west (76%). The frequencies of isolation and of fluconazole resistance among C. glabrata BSI isolates were higher in the present study (years 2001 to 2007) than in a previous study conducted from 1992 to 2001. Whereas the frequency of C. glabrata increased with patient age, the rate of fluconazole resistance declined. The oldest age group (≥80 years) had the highest proportion of BSI isolates that were C. glabrata (32%) and the lowest rate of fluconazole resistance (5%).Candidemia is without question the most important of the invasive mycoses (6, 33, 35, 61, 65, 68, 78, 86, 88). Treatment of candidemia over the past 20 years has been enhanced considerably by the introduction of fluconazole in 1990 (7, 10, 15, 28, 29, 31, 40, 56-58, 61, 86, 90). Because of its widespread usage, concern about the development of fluconazole resistance among Candida spp. abounds (2, 6, 14, 32, 47, 53, 55, 56, 59, 60, 62, 80, 86). Despite these concerns, fluconazole resistance is relatively uncommon among most species of Candida causing bloodstream infections (BSI) (5, 6, 22, 24, 33, 42, 54, 56, 65, 68, 71, 86). The exception to this statement is Candida glabrata, of which more than 10% of BSI isolates may be highly resistant (MIC ≥ 64 μg/ml) to fluconazole (6, 9, 15, 23, 30, 32, 36, 63-65, 71, 87, 91). Suboptimal fluconazole dosing practices (low dose [<400 mg/day] and poor indications) may lead to an increased frequency of isolation of C. glabrata as an etiological agent of candidemia in hospitalized patients (6, 17, 29, 32, 35, 41, 47, 55, 60, 68, 85) and to increased fluconazole (and other azole) resistance secondary to induction of CDR efflux pumps (2, 11, 13, 16, 43, 47, 50, 55, 69, 77, 83, 84) and may adversely affect the survival of treated patients (7, 10, 29, 40, 59, 90). Among the various Candida species, C. glabrata alone has increased as a cause of BSI in U.S. intensive care units since 1993 (89). Within the United States, the proportion of fungemias due to C. glabrata has been shown to vary from 11% to 37% across the different regions (west, midwest, northeast, and south) of the country (63, 65) and from <10% to >30% within single institutions over the course of several years (9, 48). It has been shown that the prevalence of C. glabrata as a cause of BSI is potentially related to many disparate factors in addition to fluconazole exposure, including geographic characteristics (3, 6, 63-65, 71, 88), patient age (5, 6, 25, 35, 41, 42, 48, 63, 82, 92), and other characteristics of the patient population studied (1, 32, 35, 51). Because C. glabrata is relatively resistant to fluconazole, the frequency with which it causes BSI has important implications for therapy (21, 29, 32, 40, 41, 45, 56, 57, 59, 80, 81, 86, 90).Previously, we examined the susceptibilities to fluconazole of 559 BSI isolates of C. glabrata and grouped the isolates by patient age and geographic location within the United States over the time period from 1992 to 2001 (63). In the present study we build upon this experience and report the fluconazole susceptibilities of 642 BSI isolates of C. glabrata collected from sentinel surveillance sites throughout the United States for the time period from 2001 through 2007 and stratify the results by geographic region and patient age. The activities of voriconazole and the echinocandins against this contemporary collection of C. glabrata isolates are also reported.     

Selection of Polymorphic Peptides from GRA6 and GRA7 Sequences of Toxoplasma gondii Strains To Be Used in Serotyping

The evaluation of Toxoplasma gondii isolates obtained from geographical environments other than Europe and North America revealed the existence of atypical strains that are not included in the three archetypal clonal lineages (lineages I, II, and III). GRA6 and GRA7 are polymorphic genes that have been used for the genotyping of Toxoplasma. The coding regions of GRA6 and GRA7 from 49 nonarchetypal strains were sequenced and compared with the sequences of type I, II, and III reference strains. Eighteen and 10 different amino acid sequences were found for GRA6 and GRA7, respectively. The polymorphisms found between the different sequences were analyzed, with the objective of defining peptides to be used for the serotyping of Toxoplasma infections. Two peptides specific for clonal lineages I and III (peptides GRA7I and GRA7III, respectively) were selected from the GRA7 locus. Three peptides specific for some atypical strains (peptides Am6, Af6, and Am7) were selected from both the GRA6 and the GRA7 loci. Serum samples from humans infected with Toxoplasma strains of known genotypes were serotyped with the selected peptides. Peptide GRA7III seems to be a good candidate for the serotyping of infections caused by type III strains. Peptide GRA7I had a very low sensitivity. Peptides Am6 and Af6 had low specificities, since they reacted with serum samples from patients infected with strains belonging to the three archetypal lineages. Although peptide Am7 was specific, it had low sensitivity.The vast majority of Toxoplasma gondii isolates from human patients and domestic animals in Europe and North America belong to three archetypal clonal lineages, namely, types I, II, and III (1, 11, 30). However, nonarchetypal strains with atypical genotypes have recently been described in unusual hosts such as sea otters (10, 42, 43) and in tropical areas such as South America and Africa (2, 32, 39, 46, 54). Genotyping studies that distinguish different types of strains are important to gain knowledge of the biodiversity of the parasite in order to understand the molecular epidemiology of Toxoplasma and to highlight the correlation between the genotype of the parasite and the pathogenesis of human toxoplasmosis.The dense granules (GRA) are parasitic organelles involved in cell invasion and in the intracellular survival of the parasite. GRA proteins are expressed by the three stages of T. gondii: the tachyzoite, bradyzoite (38), and sporozoite (55) stages. GRA6 is a GRA antigen of 32 kDa described for the first time by Lecordier et al. (38). In extracellular parasites, GRA6 exists in dense secretor granules mostly as soluble proteins. Like the other GRA proteins, GRA6 is involved in host cell invasion. GRA6 is a glycine-rich protein and behaves like an integral membrane protein within the parasitophorous vacuole (36, 41). GRA6 is considered a good marker of acute infection (27, 28, 52). However, the immune response to GRA6 is very heterogeneous (25).GRA7 is a GRA antigen of 29 kDa (26, 31). Like GRA6, it is involved in host cell invasion. This protein is associated with the parasite membrane complex, the tubular elements of the intravacuolar network, and the parasitophorous vacuolar membrane. It migrates from the GRA to the parasitophorous vacuolar membrane through the intravacuolar network during host cell invasion (9). GRA7 is an antigen characteristic of the acute phase of the infection (27, 49, 51) and a target antigen in the intracerebral immune response during the chronic phase of infection (23, 45). These immunogenic properties make this antigen a good marker for serodiagnostic studies (5, 6, 7, 50).GRA6 and GRA7 are polymorphic loci. The coding region of the GRA6 locus has been used as a marker for the genotyping of Toxoplasma (8, 15-20, 24, 33, 34, 40, 43, 47, 53). Sequencing of GRA6 detected a high degree of polymorphism (24, 57). Single nucleotide polymorphisms (SNP) in the region encoding GRA6 can be detected by methods based on PCR followed by restriction fragment length polymorphism (PCR-RFLP): digestion of the amplification products with a single endonuclease (MseI) can differentiate genotypes I, II, and III (24). Another method based on GRA6 polymorphisms is pyrosequencing (21). This technique allows the analysis of short DNA sequences and SNP. Two SNP located at positions 162 and 171 of the GRA6 gene allow the differentiation of types I, II, and III. GRA7 has been less explored as a marker for the genotyping of Toxoplasma. Preliminary results (I. Villena, unpublished data) showed that GRA7 allows the discrimination between genotypes I, II, and III and some atypical strains by PCR-RFLP.Kong et al. (35) first proposed the use of the GRA6 and GRA7 proteins for serotyping. Basically, serotyping consists of a serological test with polymorphic peptides from Toxoplasma immunogens to detect strain-specific antibodies. Different peptides were proposed on the basis of the sequences of these antigens (35, 48, 53). However, the available peptides can differentiate only type II from non-type II infections. Infections due to nonarchetypal strains are misclassified as type II strains or type I or III strains, since GRA6 C-terminal peptides specific for type II and type I or III strains cross-react with serum samples from patients with infections caused by nonarchetypal strains (53).We proposed to determine polymorphic regions of GRA6 and GRA7 with the objective of defining possible polymorphic peptides that could be used to distinguish type I from type III infections and infections due to atypical strains by serotyping. Some of the defined peptides were tested in order to evaluate their utility as serotyping markers.     

Hag Mediates Adherence of Moraxella catarrhalis to Ciliated Human Airway Cells

Moraxella catarrhalis is a human pathogen causing otitis media in infants and respiratory infections in adults, particularly patients with chronic obstructive pulmonary disease. The surface protein Hag (also designated MID) has previously been shown to be a key adherence factor for several epithelial cell lines relevant to pathogenesis by M. catarrhalis, including NCIH292 lung cells, middle ear cells, and A549 type II pneumocytes. In this study, we demonstrate that Hag mediates adherence to air-liquid interface cultures of normal human bronchial epithelium (NHBE) exhibiting mucociliary activity. Immunofluorescent staining and laser scanning confocal microscopy experiments demonstrated that the M. catarrhalis wild-type isolates O35E, O12E, TTA37, V1171, and McGHS1 bind principally to ciliated NHBE cells and that their corresponding hag mutant strains no longer associate with cilia. The hag gene product of M. catarrhalis isolate O35E was expressed in the heterologous genetic background of a nonadherent Haemophilus influenzae strain, and quantitative assays revealed that the adherence of these recombinant bacteria to NHBE cultures was increased 27-fold. These experiments conclusively demonstrate that the hag gene product is responsible for the previously unidentified tropism of M. catarrhalis for ciliated NHBE cells.Moraxella catarrhalis is a gram-negative pathogen of the middle ear and lower respiratory tract (29, 40, 51, 52, 69, 78). The organism is responsible for ∼15% of bacterial otitis media cases in children and up to 10% of infectious exacerbations in patients with chronic obstructive pulmonary disease (COPD). The cost of treating these ailments places a large financial burden on the health care system, adding up to well over $10 billion per annum in the United States alone (29, 40, 52, 95, 97). In recent years, M. catarrhalis has also been increasingly associated with infections such as bronchitis, conjunctivitis, sinusitis, bacteremia, pneumonia, meningitis, pericarditis, and endocarditis (3, 12, 13, 17-19, 24, 25, 27, 51, 67, 70, 72, 92, 99, 102-104). Therefore, the organism is emerging as an important health problem.M. catarrhalis infections are a matter of concern due to high carriage rates in children, the lack of a preventative vaccine, and the rapid emergence of antibiotic resistance in clinical isolates. Virtually all M. catarrhalis strains are resistant to β-lactams (34, 47, 48, 50, 53, 65, 81, 84). The genes specifying this resistance appear to be gram positive in origin (14, 15), suggesting that the organism could acquire genes conferring resistance to other antibiotics via horizontal transfer. Carriage rates as high as 81.6% have been reported for children (39, 104). In one study, Faden and colleagues analyzed the nasopharynx of 120 children over a 2-year period and showed that 77.5% of these patients became colonized by M. catarrhalis (35). These investigators also observed a direct relationship between the development of otitis media and the frequency of colonization. This high carriage rate, coupled with the emergence of antibiotic resistance, suggests that M. catarrhalis infections may become more prevalent and difficult to treat. This emphasizes the need to study pathogenesis by this bacterium in order to identify vaccine candidates and new targets for therapeutic approaches.One key aspect of pathogenesis by most infectious agents is adherence to mucosal surfaces, because it leads to colonization of the host (11, 16, 83, 93). Crucial to this process are surface proteins termed adhesins, which mediate the binding of microorganisms to human cells and are potential targets for vaccine development. M. catarrhalis has been shown to express several adhesins, namely UspA1 (20, 21, 59, 60, 77, 98), UspA2H (59, 75), Hag (also designated MID) (22, 23, 37, 42, 66), OMPCD (4, 41), McaP (61, 100), and a type 4 pilus (63, 64), as well as the filamentous hemagglutinin-like proteins MhaB1, MhaB2, MchA1, and MchA2 (7, 79). Each of these adhesins was characterized by demonstrating a decrease in the adherence of mutant strains to a variety of human-derived epithelial cell lines, including A549 type II pneumocytes and Chang conjunctival, NCIH292 lung mucoepidermoid, HEp2 laryngeal, and 16HBE14o-polarized bronchial cells. Although all of these cell types are relevant to the diseases caused by M. catarrhalis, they lack important aspects of the pathogen-targeted mucosa, such as the features of cilia and mucociliary activity. The ciliated cells of the respiratory tract and other mucosal membranes keep secretions moving out of the body so as to assist in preventing colonization by invading microbial pathogens (10, 26, 71, 91). Given this critical role in host defense, it is interesting to note that a few bacterial pathogens target ciliated cells for adherence, including Actinobacillus pleuropneumoniae (32), Pseudomonas aeruginosa (38, 108), Mycoplasma pneumoniae (58), Mycoplasma hyopneumoniae (44, 45), and Bordetella species (5, 62, 85, 101).In the present study, M. catarrhalis is shown to specifically bind to ciliated cells of a normal human bronchial epithelium (NHBE) culture exhibiting mucociliary activity. This tropism was found to be conserved among isolates, and analysis of mutants revealed a direct role for the adhesin Hag in binding to ciliated airway cells.     

Shiga Toxin,Cytolethal Distending Toxin,and Hemolysin Repertoires in Clinical Escherichia coli O91 Isolates

Shiga toxin (Stx)-producing Escherichia coli (STEC) strains of serogroup O91 are the most common human pathogenic eae-negative STEC strains. To facilitate diagnosis and subtyping of these pathogens, we genotypically and phenotypically characterized 100 clinical STEC O91 isolates. Motile strains expressed flagellar antigens H8 (1 strain), H10 (2 strains), H14 (52 strains), and H21 (20 strains) or were H nontypeable (Hnt) (10 strains); 15 strains were nonmotile. All nonmotile and Hnt strains possessed the fliC gene encoding the flagellin subunit of the H14 antigen (fliC_H14). Most STEC O91 strains possessed enterohemorrhagic E. coli hlyA and expressed an enterohemolytic phenotype. Among seven stx alleles identified, stx_2dact, encoding mucus- and elastase-activatable Stx2d, was present solely in STEC O91:H21, whereas most strains of the other serotypes possessed stx₁. Moreover, only STEC O91:H21 possessed the cdt-V cluster, encoding cytolethal distending toxin V; the toxin was regularly expressed and was lethal to human microvascular endothelial cells. Infection with STEC O91:H21 was associated with hemolytic-uremic syndrome (P = 0.0015), whereas strains of the other serotypes originated mostly in patients with nonbloody diarrhea. We conclude that STEC O91 clinical isolates belong to at least four lineages that differ by H antigens/fliC types, stx genotypes, and non-stx putative virulence factors, with accumulation of virulence determinants in the O91:H21 lineage. Isolation of STEC O91 from patients'' stools on enterohemolysin agar and the rapid initial subtyping of these isolates using fliC genotyping facilitate the identification of these emerging pathogens in clinical and epidemiological studies and enable prediction of the risk of a severe clinical outcome.Shiga toxin (Stx)-producing Escherichia coli (STEC) strains cause diarrhea and a life-threatening hemolytic-uremic syndrome (HUS) worldwide (23, 44). STEC strains isolated from patients usually possess, in addition to one or more stx genes, the eae gene, encoding adhesin intimin (7, 11, 16, 25, 26, 41, 49). However, a subset of STEC strains associated with human disease lack eae, and among these, strains of serogroup O91 are the most common (2, 7, 35, 37, 47, 48). In Germany during the last 5 years, serogroup O91 accounted for 6.4% to 11.0% of all STEC strains reported from human infections and was therefore the fourth-most-common STEC serogroup (after O157, O26, and O103) isolated (47, 48; http://www.rki.de). However, in contrast to eae-positive STEC strains of the three leading serogroups, which cause disease mostly in young children (47), STEC O91 is the most common serogroup isolated from adult patients (48).Despite their association with human diseases worldwide (7, 9, 11, 13, 14, 30, 35, 37, 38, 40, 47, 48), the spectrum of serotypes of STEC O91 isolates from patients and the pathogenic traits of such strains are poorly understood. Moreover, characteristics of STEC O91 strains which could assist with their isolation from human stools and further subtyping in clinical microbiological laboratories have not been systematically investigated or reported. To gain insight into the serotype composition and putative virulence factors of STEC O91 strains causing human disease and to identify characteristics which can facilitate laboratory diagnosis of these organisms, we determined the motility and flagellar phenotypes, fliC types, stx genotypes, non-stx putative virulence loci, and diagnostically useful phenotypes of 100 clinical STEC O91 isolates. Moreover, we investigated possible associations between bacterial characteristics and clinical infection phenotypes.     

Plasmid-Borne Virulence-Associated Genes Have a Conserved Organization in Virulent Strains of Avian Pathogenic Escherichia coli

Avian pathogenic Escherichia coli (APEC) is an important respiratory pathogen of poultry. Various virulence factors are responsible for determining the pathogenicity of these strains, and it is commonly believed they are encoded on large plasmids the strains carry. This study examined a series of strains, the pathogenicity of which had previously been determined by aerosol exposure, for possession of large plasmids and found all isolates carried at least one large plasmid, regardless of the level of virulence. Virulence-associated genes carried on these plasmids were also examined, and it was shown that highly virulent strains carried at least four virulence-associated genes on their largest plasmid. Two of the virulence-associated genes were shown to be chromosomally located in a strain of intermediate virulence, while no virulence-associated genes were carried by the low-virulence strain. The organization of the virulence-associated genes was shown to be highly conserved among APEC isolates of high virulence, supporting the concept of a conserved portion of the putative virulence region that contributes to the pathogenicity of APEC strains.Avian pathogenic Escherichia coli (APEC) strains cause respiratory disease and septicemia in poultry and are economically important worldwide, causing significant mortality (13). The carriage of large plasmids is considered characteristic of APEC isolates (8), and pathogenicity is thought to be determined by virulence-associated factors encoded by them (15). These factors include serum resistance, encoded by the iss gene (14), temperature-sensitive hemagglutination, encoded by tsh (10), adhesins, the production of colicin V (ColV) and the possession of iron-scavenging mechanisms, such as aerobactin production (encoded by the iucABCD operon), and the more recently identified putative iron transport system encoded by the etsABC operon (18).Another iron acquisition system found in APEC utilizes salmochelin, a catecholate siderophore. The chromosomal iroA gene cluster that encodes this system was first found in Salmonella enterica (2) and is absent from the corresponding region of the E. coli chromosome (32), although it has been found on a transmissible plasmid from a uropathogenic E. coli isolate (34). The iroA gene cluster has been found on multiple APEC virulence plasmids (9, 17, 18, 37), and deletion studies have shown that the iroA gene cluster is required for full virulence (9).A further iron transport system, designated the sitABCD system, was first identified on a pathogenicity island in Salmonella enterica serovar Typhimurium (39), and it has been shown that sitABCD is required for full virulence of Salmonella serovar Typhimurium (16). Genomic subtraction identified the plasmid-located sitA gene from the sitABCD operon as unique to an APEC strain (32), and the sitA gene was found to be more prevalent in APEC than in commensal E. coli (18, 29, 32).The sitABCD operon occurs on APEC virulence plasmids (17, 18, 30, 37), but a sitABCD deletion mutant was still pathogenic for birds, suggesting that other iron transport systems are able to compensate for the loss of sitABCD (30).The carriage of ColV plasmids has previously been thought to be essential for virulence (3, 33, 38). However, other studies have suggested it is not the presence of the ColV gene itself but other genes that these plasmids carry that are responsible for virulence (28, 35). The well-characterized APEC virulence plasmids pAPEC-O2-ColV (18) and pAPEC-1 (9) encode ColV, while carriage of the Australian APEC virulence plasmid pVM01 does not confer production of ColV (12). Despite various ColV statuses, all three of these virulence plasmids are F-type plasmids, and hence this is potentially another way to characterize APEC virulence plasmids.SopA and SopB, which have similarity to the ParA and ParB proteins of the P1 plasmid, are thought to be essential for F-plasmid partitioning (22, 24). Detection of the genes of the sopABC locus could thus indicate the presence of a putative virulence plasmid.Strain E3 is an O-nontypeable:H28 APEC field isolate (11) that carries the 151-kb virulence plasmid pVM01 (12), which contains a virulence region with the virulence-associated genes iucA, tsh, iss, iroN, and sitA, as well as hlyF, ompT, and the etsABC operon (37). The arrangement of the virulence-associated genes around pVM01 (37) is similar to that in the plasmids pAPEC-O2-ColV from APEC strain O2 (18), pAPEC-O1-ColBM from APEC strain O1 (17), and pAPEC-1 from APEC strain χ7122 (23). Identifying a specific region that is conserved in highly virulent APEC strains will facilitate diagnosis of colibacillosis by differentiation of pathogenic strains from commensal E. coli and will also enable surveillance for pathogenic isolates in the environment of poultry.This study examined six E. coli strains, some of which were isolated from diseased birds and some of which were recovered from healthy birds (11, 36). The pathogenicity of these strains has been determined using aerosol exposure (11, 36), making this the largest known collection of APEC strains fulfilling Koch''s postulates. The series of strains includes the highly virulent strains E3, E30, and E956 and the less-virulent strains E133, E1043, and E1292. The presence of the virulence-associated genes iucA, tsh, and iss in these strains has previously been elucidated by PCR amplification (36). However, while previous studies have found many of these virulence factors to be encoded by APEC strains associated with disease (29) and have suggested that they are encoded on virulence plasmids (18), they have not conclusively determined whether they are encoded on virulence plasmids or are chromosomally encoded. Similarly, although previous studies suggest that these virulence-associated genes are consistently present in isolates from diseased birds (1, 6, 18, 21, 26, 29), no study has yet determined if these genes are consistently associated with each other.The aim of this study was to examine a series of strains of known pathogenicities for the possession of large plasmids and to determine if known virulence-associated genes from the putative virulence region were carried on them. The second objective was to investigate any association between the virulence-associated genes.     

Identification of a Variety of Staphylococcus Species by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry

Whole-cell fingerprinting by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) in combination with a dedicated bioinformatic software tool (MALDI Biotyper 2.0) was used to identify 152 staphylococcal strains corresponding to 22 staphylococcal species. Spectra of the 152 isolates, previously identified at the species level using a sodA gene-based oligonucleotide array, were analyzed against the main spectra of 3,030 microorganisms. A total of 151 strains out of 152 (99.3%) were correctly identified at the species level; only one strain was identified at the genus level. The MALDI-TOF MS method revealed different clonal lineages of Staphylococcus epidermidis that were of either human or environmental origin, which suggests that the MALDI-TOF MS method could be useful in the profiling of staphylococcal strains. The topology of the dendrogram generated by the MALDI Biotyper 2.0 software from the spectra of 120 Staphylococcus reference strains (representing 36 species) was in general agreement with that inferred from the 16S rRNA gene-based analysis. Our findings indicate that the MALDI-TOF MS technology, associated with a broad-spectrum reference database, is an effective tool for the swift and reliable identification of Staphylococci.Most staphylococci are harmless and reside normally on the skin and mucous membranes of humans and other organisms (16, 22, 34). Staphylococcal strains are isolated from various food products in which they are involved in fermentation (18, 29). Staphylococcus species can cause a wide variety of diseases in humans and other animals (2, 22, 30-32, 35). S. aureus is a major pathogen in human infections (31). Several other Staphylococcus species have also been implicated in human infections, notably S. saprophyticus, S. epidermidis, S. lugdunensis, and S. schleiferi (4, 16, 31, 34). Coagulase-negative staphylococci (CoNS) have emerged as predominant pathogens in hospital-acquired infections (4, 16, 31, 34). One of the major challenges of daily diagnostic work is therefore to identify Staphylococcus species.Several manual and automated methods based on phenotypic characteristics have been developed for the identification of Staphylococci (12, 24). Unfortunately, these systems have their limitations, mostly due to phenotypic differences between strains from the same species (6, 10, 19, 21). Over the last 10 years, many genotypic methods based on the analysis of selected DNA targets have been designed for species-level identification of most common isolated CoNS (20, 26, 33). The sequence polymorphism of the sodA gene has significant discriminatory power (20) and allows the development of assays based on DNA chip technologies (“Staph array”) (8). Recently, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) using protein “fingerprints” was used for the identification of microorganisms (1, 3, 5, 9, 11, 14, 25, 36). In the present study, we assessed the ability of the MALDI Biotyper system (Bruker Daltonique, Wissembourg, France) to identify Staphylococcus species of clinical and environmental origins previously identified by sodA gene-based oligonucleotide array (8).     

Genetic and antigenic diversities of major immunoreactive proteins in globally distributed Ehrlichia canis strains

The extent of knowledge regarding the diversity of globally distributed Ehrlichia canis strains has been limited to information gained from a few evolutionarily conserved genes. In this study, E. canis strains from the United States (strain Jake [US]), Brazil (strain São Paulo [BR]), and Israel (strain 611 [IS] and Ranana [IS-R]) were used to examine the antigenic and genetic diversities of four well-characterized major immunoreactive protein genes/proteins. gp36 and gp200 were the most divergent genes, and nucleotide substitutions in the gp36 tandem repeat region of the IS strain, but not the IS-R strain, resulted in two amino acid differences (S→P and P→T) in each nine-amino-acid repeat (epitope-containing region). DNA sequences of gp19 and gp140 were completely conserved in the US and BR strains, but differences were found in the Israeli strains, including two fewer tandem repeats in gp140 and a single amino acid substitution in gp19 from the IS strain. E. canis whole-cell lysates from each isolate were examined by Western immunoblotting using sera from naturally infected dogs from each country, and four major immunoreactive proteins (gp19, gp36, gp140, and gp200) were identified in each strain using protein-specific antisera. The US and BR strains exhibited highly conserved immunoreactive protein profiles, while some differences were identified in the IS strain. Sera from naturally infected Israeli dogs confirmed gene sequencing information, which demonstrated two distinct E. canis strains, defined by the gp36 gene. Conversely, gp19 was strongly reactive and present in all E. canis isolates. gp140 and gp200 were also present in all strains, although gp140 in the IS strain had two fewer tandem repeats and exhibited a smaller mass.Ehrlichia canis is a globally distributed, tick-transmitted, obligately intracellular bacterium that is the primary etiological agent of canine monocytic ehrlichiosis and has been identified as being the cause of human ehrlichiosis in patients from Venezuela (38, 39). Rickettsiosis in dogs caused by E. canis was first reported in 1935 in Algeria and was later reported in southern India and other parts of Africa in the 1940s (9, 31). Subsequently, E. canis was relatively unrecognized until it was associated with outbreaks of canine tropical pancytopenia in Singapore and Malaysia from 1963 to 1968 (51) and was identified as being the cause of an epizootic of canine tropical pancytopenia in U.S. military dogs stationed in Vietnam in late 1968 (17, 36). E. canis infections have since been well documented in the United States, Israel, Brazil, and Vietnam (1, 3, 12, 16, 20-22, 36, 49), and serologic and/or molecular evidence of infection in temperate regions where Rhipicephalus sanguineus is commonly found, including Central and South America, the Caribbean, parts of Africa, southern Europe, and southeast Asia, has also been reported (2, 5-8, 15, 18, 19, 23, 32, 33, 41, 42, 44, 50).The development of globally useful serologically and molecularly based diagnostics as well as effective vaccines for canine monocytic ehrlichiosis is dependent on an understanding of the genetic diversity of E. canis, particularly with respect to major immunoreactive proteins. Molecular characterization of evolutionarily conserved genes such as 16S rRNA has provided little information on strain diversity and suggests a high level of conservation (39, 40, 43, 47, 48). Similarly, the immunoreactive major outer membrane proteins p28 and p30 in U.S. and Venezuelan strains of E. canis appear to be highly conserved (13, 29, 30, 46), an observation that was extended to characterized E. canis strains from six human patients from Venezuela (38). Other genes such as the thio-oxidoreductase gene (dsb) and gltA were also found to be conserved in geographically dispersed strains (23, 32).The genome of E. canis has been sequenced, and a small group of acidic tandem repeat- and ankyrin repeat-containing proteins associated with host-pathogen interactions were identified (24). Several of these proteins are considered major immunoreactive proteins and have been well studied, including gp200, gp140, gp36, and gp19 (11, 25, 26, 28, 53). E. canis gp36 is an acidic serine-rich protein that contains a major antibody epitope in the tandem repeat region (11). Examination of the gp36 gene in U.S., Brazilian, and Cameroonian strains of E. canis identified variations in the numbers of tandem repeats and nucleic acid changes that resulted in four amino acid substitutions (10). However, the diversities of other major immunoreactive E. canis proteins in globally dispersed strains are not known. A homogeneous pattern of proteins reacting with E. canis dog sera from the United States, France, Israel, and the Virgin Islands by immunoblotting was previously reported (14). However, differences in protein reactivity were noted with sera collected from dogs from Italy and Zimbabwe, suggesting the potential for diversity in the antigenic composition of E. canis strains in these countries (14).The objective of this study was to determine the genetic and antigenic diversities of proteins subject to immune pressure in globally dispersed strains of E. canis. Four major immunoreactive protein genes (gp200, gp140, gp36, and gp19) were sequenced from each strain, and immunoblotting profiles for E. canis whole-cell lysates were compared. Strains from the United States and Brazil exhibited homogeneous immunoblotting patterns compared to that of the strain from Israel. Sequencing of four major immunoreactive protein genes demonstrated that U.S. and Brazilian strains were highly similar and that strains from Israel were the more divergent.     

Acanthamoeba culbertsoni Elicits Soluble Factors That Exert Anti-Microglial Cell Activity

Acanthamoeba culbertsoni is an opportunistic pathogen that causes granulomatous amoebic encephalitis (GAE), a chronic and often fatal disease of the central nervous system (CNS). A hallmark of GAE is the formation of granulomas around the amoebae. These cellular aggregates consist of microglia, macrophages, lymphocytes, and neutrophils, which produce a myriad of proinflammatory soluble factors. In the present study, it is demonstrated that A. culbertsoni secretes serine peptidases that degrade chemokines and cytokines produced by a mouse microglial cell line (BV-2 cells). Furthermore, soluble factors present in cocultures of A. culbertsoni and BV-2 cells, as well as in cocultures of A. culbertsoni and primary neonatal rat cerebral cortex microglia, induced apoptosis of these macrophage-like cells. Collectively, the results indicate that A. culbertsoni can apply a multiplicity of cell contact-independent modes to target macrophage-like cells that exert antiamoeba activities in the CNS.Acanthamoeba culbertsoni belongs to a group of free-living amoebae, such as Balamuthia mandrillaris, Naegleria fowleri, and Sappinia pedata, that can cause disease in humans (46, 56). Acanthamoeba spp. are found worldwide and have been isolated from a variety of environmental sources, including air, soil, dust, tap water, freshwater, seawater, swimming pools, air conditioning units, and contaminated contact lenses (30). Trophozoites feed on bacteria and algae and represent the infective form (47, 56). However, under unfavorable environmental conditions, such as extreme changes in temperature or pH, trophozoites transform into a double-walled, round cyst (22, 45).Acanthamoeba spp. cause an infection of the eye known as amoebic keratitis (AK), an infection of the skin referred to as cutaneous acanthamoebiasis, and a chronic and slowly progressing disease of the central nervous system (CNS) known as granulomatous amoebic encephalitis (GAE) (22, 23, 30, 56). GAE is most prevalent in humans who are immunocompromised (30, 33, 40) and has been reported to occur among individuals infected with the human immunodeficiency virus (HIV) (28). It has been proposed that Acanthamoeba trophozoites access the CNS by passage through the olfactory neuroepithelium (32) or by hematogenous spread from a primary nonneuronal site of infection (23, 24, 33, 53).In immune-competent individuals, GAE is characterized by the formation of granulomas. These cellular aggregates consist of microglia, macrophages, polymorphonuclear cells, T lymphocytes, and B lymphocytes (24, 30). The concerted action of these immune cells results in sequestration of amoebae and is instrumental in slowing the progression of GAE. This outcome is consistent with the observation that granulomas are rarely observed in immunocompromised individuals (34) and in mice with experimentally induced immune suppression following treatment with the cannabinoid delta-9-tetrahydrocannabinol (Δ⁹-THC) (8).Microglia are a resident population of macrophages in the CNS. These cells, along with CNS-invading peripheral macrophages, appear to play a critical early effector role in the control of Acanthamoeba spread during GAE (4, 5, 29, 31). In vitro, microglia have been shown to produce an array of chemokines and cytokines in response to Acanthamoeba (31, 51). However, these factors appear not to have a deleterious effect on these amoebae (29).Acanthamoeba spp. produce serine peptidases, cysteine peptidases, and metallopeptidases (1, 2, 9, 10, 14, 16, 18, 19, 21, 25, 26, 37, 38, 41, 42, 52). In the present study, it is demonstrated that serine peptidases secreted by A. culbertsoni degrade chemokines and cytokines that are produced by immortalized mouse BV-2 microglia-like cells. In addition, soluble factors present in cocultures of A. culbertsoni and BV-2 cells induced apoptosis of the BV-2 cells. Collectively, these results suggest a mode through which A. culbertsoni can evade immune responsiveness in the CNS.     

Virulence and Cellular Interactions of Burkholderia multivorans in Chronic Granulomatous Disease

Chronic granulomatous disease (CGD) patients are susceptible to life-threatening infections by the Burkholderia cepacia complex. We used leukocytes from CGD and healthy donors and compared cell association, invasion, and cytokine induction by Burkholderia multivorans strains. A CGD isolate, CGD1, showed higher cell association than that of an environmental isolate, Env1, which correlated with cell entry. All B. multivorans strains associated significantly more with cells from CGD patients than with those from healthy donors. Similar findings were observed with another CGD pathogen, Serratia marcescens, but not with Escherichia coli. In a mouse model of CGD, strain CGD1 was virulent while Env1 was avirulent. B. multivorans organisms were found in the spleens of CGD1-infected mice at levels that were 1,000 times higher than those found in Env1-infected mice, which was coincident with higher levels of the proinflammatory cytokine interleukin-1β. Taken together, these results may shed light on the unique susceptibility of CGD patients to specific pathogens.Chronic granulomatous disease (CGD) is a rare primary immunodeficiency resulting from genetic defects in the phagocyte NAPDH oxidase. It is characterized by life-threatening infections caused by specific bacteria and fungi, leading to pneumonias, tissue abscesses, and exuberant granuloma formation (38). The Burkholderia cepacia complex (Bcc) includes at least 10 distinct species and is a leading cause of bacterial infections in CGD (44). Patients with cystic fibrosis (CF) also develop Bcc infections with various outcomes, ranging from no change in clinical course to a more rapid deterioration of lung function to the dreadful cepacia syndrome, which is characterized by necrotizing pneumonia and sepsis (25, 45). Interestingly, Bcc rarely causes infection in healthy individuals, but it can infect patients undergoing bronchoscopies and other procedures (4).Within the Bcc, Burkholderia cenocepacia and Burkholderia multivorans are commonly isolated from CF and non-CF patients (4, 32); the rate of B. multivorans infection now exceeds that of B. cenocepacia at several CF centers (15). In contrast to the high transmissibility of some CF B. cenocepacia strains (i.e., the epidemic lineage ET12) (24, 25), CF B. multivorans infections likely reflect independent acquisitions from unrelated sources (24). Curiously, unlike B. cenocepacia, B. multivorans has been recovered from environmental samples only rarely (1, 24), and it is the most frequently found species among CGD patients (16, 17).The mechanisms by which the Bcc causes disease specifically in CF are not known. Bcc isolates can survive within macrophages (28, 33) and respiratory epithelial cells (5, 21) and can invade epithelial cells in vivo (8, 10) and persist in the lung (9, 10). Cell infection assays using monocytes, macrophages, and epithelial cells (10, 11, 29, 46) show great variability among individual Bcc strains, with no clear correlation between those isolated from CF patients and those isolated from the environment (22). For the most part, these studies have been carried out using tissue culture models (28, 29, 43) and, in some cases, CF human or CF mouse cell systems (34, 35).Much less is known about the interaction between the Bcc and CGD despite the availability of animal models for the disease (20, 31). B. cenocepacia induced the necrosis of human CGD neutrophils but not normal controls (6). Similarly to healthy people, normal mice are resistant to the Bcc and usually show only transient infections upon inoculation (8, 37). On the other hand, CGD mice are highly susceptible to Bcc infection and show clinical signs that are similar to those of the human disease (20, 31, 37).To address why B. multivorans is a pathogen in CGD, we initiated studies with strains isolated from CGD patients and CGD cells. Strains of B. multivorans differed in cell association and cell entry. We found a preferential association of bacteria with CGD instead of normal leukocytes as shown by microscopy and culture techniques. This preferential association is shared by another CGD pathogen, Serratia marcescens, but not by Escherichia coli. Finally, we demonstrate dramatic differences in virulence in B. multivorans strains in a mouse model of CGD.     

Avian-Pathogenic Escherichia coli Strains Are Similar to Neonatal Meningitis E. coli Strains and Are Able To Cause Meningitis in the Rat Model of Human Disease

Escherichia coli strains causing avian colibacillosis and human neonatal meningitis, urinary tract infections, and septicemia are collectively known as extraintestinal pathogenic E. coli (ExPEC). Characterization of ExPEC strains using various typing techniques has shown that they harbor many similarities, despite their isolation from different host species, leading to the hypothesis that ExPEC may have zoonotic potential. The present study examined a subset of ExPEC strains: neonatal meningitis E. coli (NMEC) strains and avian-pathogenic E. coli (APEC) strains belonging to the O18 serogroup. The study found that they were not easily differentiated on the basis of multilocus sequence typing, phylogenetic typing, or carriage of large virulence plasmids. Among the APEC strains examined, one strain was found to be an outlier, based on the results of these typing methods, and demonstrated reduced virulence in murine and avian pathogenicity models. Some of the APEC strains tested in a rat model of human neonatal meningitis were able to cause meningitis, demonstrating APEC''s ability to cause disease in mammals, lending support to the hypothesis that APEC strains have zoonotic potential. In addition, some NMEC strains were able to cause avian colisepticemia, providing further support for this hypothesis. However, not all of the NMEC and APEC strains tested were able to cause disease in avian and murine hosts, despite the apparent similarities in their known virulence attributes. Thus, it appears that a subset of NMEC and APEC strains harbors zoonotic potential, while other strains do not, suggesting that unknown mechanisms underlie host specificity in some ExPEC strains.Escherichia coli strains causing extraintestinal disease are known as extraintestinal pathogenic E. coli (ExPEC) and include the uropathogenic E. coli (UPEC), neonatal meningitis E. coli (NMEC), and avian-pathogenic E. coli (APEC) subpathotypes. Recent studies have shown that members of various ExPEC subpathotypes harbor similar virulence-associated genes, despite their isolation from varied hosts and tissues (3, 8, 10, 20, 25, 27, 30, 32), and genomic sequencing of APEC O1 revealed that only 4.5% of the genome was not found in the other ExPEC strains sequenced (17). More recently, a cluster of isolates from human and avian hosts thought to represent potential zoonotic pathogens has been identified (20).Common among the isolates of this mixed cluster are genes associated with the conserved region of large virulence plasmids, which are a defining trait of the APEC subpathotype (15, 19, 24, 36, 37) and which are essential for APEC virulence (5, 23). Interestingly, a closely related plasmid that was associated with high-level bacteremia in a neonatal rat meningitis model has also been described in an NMEC isolate (30).Other virulence traits are also shared among ExPEC subpathotypes. Indeed, few traits, if any, appear to be exclusive to a particular ExPEC subpathotype, and in fact, some traits that were thought to be exclusive have been shown to contribute to the pathogenesis of more than one condition (8).Such similarities in the virulence traits found among APEC and other ExPEC subpathotypes have led to speculation that APEC has zoonotic potential (20, 25, 27) and may be a food-borne source of ExPEC causing disease in humans (10, 14, 18, 22). Indeed, ExPEC strains have been identified in retail foods and poultry products (7, 11, 12, 18), and at least one study has found avian isolates to be indistinguishable from human isolates (10). However, other studies showed that human ExPEC strains were clearly distinct from avian strains (6) and that the consumption of poultry or contact with poultry did not correlate with the colonization of antimicrobial-resistant E. coli (34).Here, we seek to further test the hypothesis that APEC strains have zoonotic potential. Of particular interest are O18 strains, which are common among human NMEC strains but which are also found among APEC strains (20, 26). In fact, it has been suggested that APEC O18:K1:H7 strains are potential human pathogens (27). Though it has been shown that human ExPEC strains can cause avian colibacillosis similar to that caused by APEC, suggesting that these ExPEC strains are not host specific (26), it has also been reported that E. coli strains from avian septicemia are more virulent to chicks than NMEC strains (33). However, the ability of APEC to cause disease in mammals has not yet been established.The aim of the present study was to explore the zoonotic potential of NMEC and APEC O18 strains by comparing their plasmid contents, genotypes, phylogenetic group assignments, pulsed-field gel electrophoresis (PFGE) patterns, and sequence types (ST), determined by multilocus sequence typing (MLST), and their abilities to cause disease in the rat model of human neonatal meningitis and chicken models of avian colisepticemia.     

Genetic Characterization of the Capsulation Locus of Haemophilus influenzae Serotype e

The capsulation (cap) locus of Haemophilus influenzae type e (Hie) was characterized and sequenced. No IS1016 element was found to flank the locus. The 18.2-kb locus included 14 open reading frames (ORFs), which were grouped into three functional regions. Eight new ORFs (named ecs1 to ecs8) were identified in the Hie capsule-specific region II.In the post-Haemophilus influenzae serotype b (Hib) vaccine era, concern about the potential emergence of non-vaccine-preventable strains has arisen (1, 17, 20, 23, 26). In encapsulated H. influenzae strains, the genes for the production of the polysaccharide capsules are organized in a capsulation (cap) locus, which consists of three different functional regions (11, 13). Regions I and III are common to all capsular types and contain genes necessary for transport and process of the capsular material, while region II contains serotype-specific biosynthesis genes (7, 10, 18, 19, 25).Invasive disease caused by H. influenzae serotype e (Hie) strains has recently been observed in Italy, suggesting the importance of further molecular investigations on Hie cap locus (4, 5). It is recognized that the Hie capsule is a copolymer of the repeat unit of an N-acetylglucosamine and N-acetylmannosamine uronic acid (22, 24), but the genes involved in the polysaccharide biosynthesis have neither been identified nor characterized.In the present study, we characterized the Hie cap locus for the first time. Eleven invasive Hie strains isolated in Italy during the period of January 2000 to December 2008 were analyzed. The strains were identified as type e by PCR capsular genotyping (6).     

Molecular Identification and Susceptibility of Trichosporon Species Isolated from Clinical Specimens in Qatar: Isolation of Trichosporon dohaense Taj-Aldeen,Meis & Boekhout sp. nov.

Trichosporon species have been reported as emerging pathogens and usually occur in severely immunocompromised patients. In the present work, 27 clinical isolates of Trichosporon species were recovered from 27 patients. The patients were not immunocompromised, except for one with acute myeloid leukemia. Sequence analysis revealed the isolation of Trichosporon dohaense Taj-Aldeen, Meis & Boekhout sp. nov., with CBS 10761^T as the holotype strain, belonging to the Ovoides clade. In the D1-D2 large-subunit rRNA gene analysis, T. dohaense is a sister species to T. coremiiforme, and in the internal transcribed spacer analysis, the species is basal to the other species of this clade. Molecular identification of the strains yielded 17 T. asahii, 3 T. inkin, 2 T. japonicum, 2 T. faecale, and 3 T. dohaense isolates. The former four species exhibited low MICs for five antifungal azoles but showed high MICs for amphotericin B. T. dohaense demonstrated the lowest amphotericin B MIC (1 mg/liter). For the majority of T. asahii isolates, amphotericin B MICs were high (MIC at which 90% of isolates were inhibited [MIC₉₀], ≥16 mg/liter), and except for fluconazole (MIC₉₀, 8 mg/liter), the azole MICs were low: MIC₉₀s were 0.5 mg/liter for itraconazole, 0.25 mg/liter for voriconazole, 0.25 mg/liter for posaconazole, and 0.125 mg/liter for isavuconazole. The echinocandins, caspofungin and anidulafungin, demonstrated no activity against Trichosporon species.Trichosporon species are yeast-like fungi, widely distributed in nature and commonly isolated from soil and other environmental sources, which have been involved in a variety of opportunistic infections and have been recognized as emerging fungal pathogens in immunocompromised hosts (19, 79, 80). Disseminated Trichosporon infections are potentially life-threatening and are often fatal in neutropenic patients (7, 22). Although uncommon, pathogenic species of this genus have been reported increasingly, mostly in patients with malignant diseases (3, 6, 9, 10, 11, 20, 32, 44, 47, 48, 63, 77), neonates (18, 56, 84), a bone marrow transplant recipient (22), a solid organ transplant recipient (50), and patients with human immunodeficiency virus (34, 35, 46). Trichosporon has also been reported to cause fungemia (5, 9, 25, 29, 30, 33, 53, 62). Members of the genus Trichosporon have occasionally been implicated as nail pathogens (16, 28, 74) and in subcutaneous infections (66). Trichosporon is considered an opportunistic agent, and therefore, recovery of Trichosporon species capable of growing at 37°C, especially from immunocompromised patients, should be regarded as potentially significant. Several reports have addressed the difficulty of identifying Trichosporon to the species level by physiological and biochemical characteristics (2, 64); therefore, molecular methods based on the sequencing of the internal transcribed spacer (ITS) have been developed (15, 69, 71, 72).In the present paper, we report the isolation of Trichosporon species from clinical specimens over a 4-year period in Qatar, the poor performance of biochemical identification methods, the significance of molecular identification, and the antifungal susceptibility data for the isolates. While investigating the molecular identification of Trichosporon species, we found three strains that do not match any of the published strains in the literature. We describe this organism as Trichosporon dohaense Taj-Aldeen, Meis & Boekhout, sp. nov., the name proposed for this species.     

Single Nucleotide Polymorphisms on the Road to Strain Differentiation in Mycobacterium ulcerans

The genomic fine-typing of strains of Mycobacterium ulcerans, the causative agent of the emerging human disease Buruli ulcer, is difficult due to the clonal population structure of geographical lineages. Although large sequence polymorphisms (LSPs) resulted in the clustering of patient isolates originating from across the globe, differentiation of strains within continents using conventional typing methods is very limited. In this study, we analyzed M. ulcerans LSP haplotype-specific insertion sequence elements among 83 M. ulcerans strains and identified single nucleotide polymorphisms (SNPs) that differentiate between regional strains. This is the first genetic discrimination based on SNPs of M. ulcerans strains from African countries where Buruli ulcer is endemic, resulting in the highest geographic resolution of genotyping so far. The findings support the concept of genome-wide SNP analyses as tools to study the epidemiology and evolution of M. ulcerans at a local level.Mycobacterium ulcerans causes the devastating cutaneous disease Buruli ulcer (BU). More than 30 countries worldwide have reported this emerging disease, reaching epidemic proportions in some areas, and children between the ages of 5 and 15 in the rural wetlands of West Africa are most affected (38). Although proximity to marshes and wetlands is a risk factor, the mode of transmission remains an enigma (10, 26, 27, 36, 37). Discrimination of genetic variants has become an indispensable tool to unravel the evolution, epidemiology, and transmission of pathogenic organisms and to gain insight into host-pathogen interactions (6, 13, 24). In M. ulcerans, such elucidation is impossible due to a remarkable lack of genetic diversity on a local geographic scale (22). Conventional genetic differentiation tools commonly used for phylogenetic profiling in Mycobacterium tuberculosis, such as restriction fragment length polymorphism, amplified fragment length polymorphism, variable-number tandem repeats (VNTR), and multilocus sequence typing, could distinguish between continental lineages only when applied to M. ulcerans (1-4, 7, 8, 15, 18, 29, 31, 34, 35). However, two publications using VNTRs reported the first discrimination of strains between and within African countries (16, 33). The identification of regions of difference (RDs) in a worldwide collection of M. ulcerans isolates led to an evolutionary scheme on the continental level, with two distinct genetic lineages that can be subgrouped into six haplotypes (20, 28). Strains of the “ancestral” lineage are genetically closer to Mycobacterium marinum, the progenitor of M. ulcerans, whereas the “classical” lineage accounts for the majority of BU cases and represents the most virulent genotype. Characterization of the large sequence polymorphisms (LSPs) showed that insertion sequence (IS) element (ISE) expansion is associated with the observed genome instability (17, 19, 40). ISs are compact mobile DNA segments capable of inserting at multiple sites in a target molecule, usually by a recombinase that is encoded by a coding sequence (CDS) contained within the ISE itself (23). Thus, uncontrolled duplications and insertions of ISEs occur at relatively high frequency in replicating bacteria, leading to genomic insertions, deletions, and rearrangements that have the potential for molecular epidemiological applications. In M. tuberculosis, until recently, IS-mediated insertions/deletions (InDels) used to be the principal source of genome plasticity (6) and are widely used as evolutionary markers in epidemiological studies. For M. ulcerans, two ISEs were defined, IS2404 and IS2606 (30, 32). Earlier, site-specific IS2404 elements were identified to be unique for and confined to distinct M. ulcerans haplotypes (19). Here, we specifically amplified such unique ISEs and compared their sequences for a collection of 83 M. ulcerans isolates including 67 derived from Africa. We aimed at the detection of single nucleotide polymorphisms (SNPs) in these ISEs that made genetic distinction within haplotypes and on a regional level possible.